1. Field of the Invention
This invention relates to the purification and use of a novel family of fibrinogenolytic proteases. Specifically, this invention relates to a fibrinogenolytic protease which possesses a strong beta-fibrinogenolytic activity and does not cause formation of fibrin clots, known effects associated with thrombin and thrombin-like proteases of snake venom. In addition, the fibrinogenolytic proteases of the present invention exhibits anti-clotting and antihypertensive effects on experimental animals.
2. Description of the Related Art
Venoms from various snake species alter the haemostatic and blood coagulation systems of human victims or experimental animals in a complex manner. Different venoms contain multiple components which behave as either pro- or anti-coagulants that directly or indirectly induce or inhibit fibrinogen and/or platelet aggregation and related complex biochemical processes, resulting in common clinical complications of blood clotting or uncontrolled hemorrhage by envenomation of snakebites [Ouyang, C. (1957) J. Formosan Med. Assoc. 56, 435-448; Meaume, J. (1966) Toxicon 4, 25-58; and Kini, R. M., and Evans, H. J. (1990) Toxicon 28, 1387-1422.] These apparently contradicting activities have been attributed to the presence of fibrinogenolytic or fibrinogen clotting enzymes in snake venoms [Brinkhous, K. M., and Smith, S. V. (1988) in Hematology, Haemostasis and Animal Venoms (Pirkle, H. and Markland, F. S., Jr., Eds.) Vol. 7, pp. 363-375, Marcel Dekker, New York; Stocker, K. F. (1990) in Medical Use of Snake Venom Proteins (Stocker, K. F., Ed.) pp. 97-160, CRC Press Boston, Mass.; and Tu, A. T. (1982) In Rattlesnake Venoms: Their Actions and Treatment (Tu, A. T., Ed.), pp. 247-312, Marcel Dekker, New York.] On the other hand, platelet-aggregating enzymes in venom generally lack fibrinogenolytic activity, but can directly aggregate platelets in platelet-rich plasma [Serrano, S. M. T., Mentele, R., Sampaio, C. A. M., and Fink, E. (1995) Biochemistry 34, 7186-7193.] Current interest is directed to some fibrinolytic proteinases including metalloproteinases and thrombin-like enzymes because of their potential clinical application in the treatment of vascular thrombotic diseases [Markland, F. S. Jr. (1998) Thromb. Haemost. 79, 668-674.]
It is well known that snake venoms contain complex mixtures of pharmacologically active peptides and proteins. Reptilian venoms, particularly those obtained from the snake families of Crotalidae and Viperidae, are also shown to possess many different fibrinogenolytic proteases which may initiate or affect blood coagulation process associated with snakebites [Tu, A. T. (1982) In Rattlesnake Venoms: Their Actions and Treatment (Tu, A. T., Ed.), pp. 247-312, Marcel Dekker, New York.] Different researchers reported disparate proteases from venoms of various crotalid snakes. They included crotalase, a thrombin-like enzyme isolated from the American-Eastern diamondback rattlesnake (Crotalus adamanteus) [Markland, F. S., and Damus, P. S. (1971) J. Biol. Chem. 246, 6460-6473], hemorrhagic toxins, anticoagulant proteases and kallikrein-like enzymes from the American-Western diamondback rattlesnake Crotalus atrox [Pandya, B. V., and Budzynski, A. Z. (1984) Biochemistry 23, 460-470; Bjarnason, J. B., Barish, A., Direnzo, G. S., Campbell, R., and Fox, J. W. (1983) J. Biol. Chem. 258, 12566-12573.] Applicant has previously evaluated the venom components from Crotalus atrox and found that all fractions isolated from the anion-exchange chromatography showed varying extents of specific proteolytic activity against alpha-and/or beta-chains of fibrinogen molecules [Chiou, S. -H., Hung, C. -C., and Lin, C. -W. (1992) Biochem. International 26, 105-112; Chiou, S. -H., Hung, C. -C., and Huang, K. -F. (1992) Biochem. Biophys. Res. Commun. 187, 389-396.] Concurrently, studies on the toxin components from Taiwan habu (Trimeresurus mucrosquamatus) [Ouyang, C., and Teng, C. M. (1976) Biochim. Biophys. Acta 420, 298-308; Huang, K. -F., Hung, C. -C., and Chiou, S. -H. (1993) Biochem. Mol. Biol. International 31, 1041-1050; and Hung, C. -C., Huang, K. -F., and Chiou, S. -H. (1994) Biochem. Biophys. Res. Commun. 205, 1707-1715.], a major and abundant crotalid species in Taiwan, indicated several kinds of fibrinogenases present in this phylogenetically related species to those American rattlesnakes.
Concerning the pharmacological action of Formosan snake venoms on blood coagulation, it was reported early in 1921-1925 that the crude venoms of two crotalid snake species, Agkistrodon acutus and Trimeresurus gramineus, had a coagulant action on whole blood and plasma, while the venom of another species Trimeresurus mucrosquamatus of the same family showed an inhibitory action Ouyang, C. (1957) J. Formosan Med. Assoc. 56, 435-448. The inhibitory action on blood coagulation was believed to be caused mostly by destruction of fibrinogen in the case of the venom of Trimeresurus mucrosquamatus. 
As one object of the present invention, applicant discloses herein a family of fibrinogenases isolated from Taiwan habu, which show a specific fibrinogen-degrading activity without being associated with any activity causing fibrin clot formation. In addition, the type of fibrinogenases posesses an unexpectedly strong kallikrein-like hypotensive activity on experimental rats and may find their clinical applications in hypertension therapy.
Another object of the present invention is to develop a new expression process to produce snake venom proteins, including those identified fibrinogenases of the present invention. Expression and purification of recombinant proteins from host organisms are often a critical and time-consuming task in achieving the goal of obtaining pure and large quantities of proteins from recombinant sources [Uhlen, M., and Moks, T. (1990) Methods Enzymol. 185, 129-143.] Facile removal of contaminant expression proteins is essential to accurate characterisation of functional properties of a cloned protein. The most common solution to this problem is to engineer the expressed protein product so as to contain additional amino acid residues which give a unique property to the protein of interest that can be exploited for purification purposes. Such a strategy would greatly increase the availability of recombinant proteins for further structural and functional study. The protein with those additional amino acid residues can generally bind to transition metal ions, thereby allowing the protein to be purified using immobilized metal ion affinity chromatography (xe2x80x9cIMACxe2x80x9d) [Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Nature 258, 598-599.] In this method a specific chelating peptide can be cloned onto the amino terminus (xe2x80x9cN-terminusxe2x80x9d) of a recombinant protein to serve as a purification tag or handle [Smith, M. C., Furman, T. C., Ingolia, T. D., and Pidgeon, C. (1988) J. Biol. Chem. 263, 7211-7215] and subsequently purified by using IMAC.
Recent advance in recombinant DNA technology has allowed in vitro fusion of genes or gene fragments in a simple and predictable manner. There are several reasons to use gene fusion for expression of recombinant proteins in heterologous hosts. In particular, a more reliable and reproducible method to obtain a native protein might be to use in vitro cleavage of the fusion protein, as compared to in vivo removal of the formyl-methionine or cleavage of a signal peptide, which in both cases may yield a heterogeneous N-terminus. Many expression vectors currently used to encode, encoding a protease cleavage site that allows release of carboxyl-terminal (xe2x80x9cC-terminalxe2x80x9d) fusion partners from fusion proteins without leaving unwanted amino-terminal (xe2x80x9cN-terminalxe2x80x9d) amino acid residues behind. In snake venom protein expression system, removal of the upstream fusion partners has been conducted with one of the three different proteases, including thrombin (Maeda, M., Satoh, S., Suzuki, S., Niwa, M., Ioth, N., and Ya-mashina, I. (1991) J. Biochem. (Tokyo) 109, 632-637; Rosenthal, J. A., Levandoski, M. M., Chang, B., Potts, J. F., Shi, Q. -L., and Hawrot, E. (1999) Biochemistry 38, 7847-7855), factor Xa (Rosenthal, J. A., Hsu, S. H., Schneider, D., Gentile, L. N., Messier, N. J., Vaslet, C. A., and Hawrot, E. (1994) J. Biol. Chem. 269, 11178-11185; Zhang, Y., Wisner, A., Maroun, R. C., Choumet, V., Xiong, Y., and Bon, C. (1997) J. Biol. Chem. 272, 20531-20537), and enterokinase (Moura-da-Silva, A. M., Linica, A., Della-Casa, M. S., Kamiguti, A. S., Ho, P. L., Crampton, J. M., and Theakston, R. D. (1999) Arch. Biochem. Biophys. 369, 295-301). However, with a serine protease (Tm-5) from Taiwan habu (Trimeresurus mucrosquamatus) (Hung, C. -C., Huang, K. -F., and Chiou, S. -H. (1994) Biochem. Biophys. Res. Commun. 205, 1707-1715), applicants repeatedly encountered difficulties in removing the attached N-terminal polyhistidine tag due to the inability of the commercial enterokinase to cleave efficiently the desired product from the expressed fusion venom protein. There is therefore a need for a different expression system for producing venom proteases.
These and other objects are attained by the present invention which provides two venom proteases showing a strong kallikrein-like hypotensive activity, potentially useful in hypertension therapy. The present invention also provides a new expression system to produce the aforementioned venom proteases using modern biotechnology.
In the present invention, two serine proteases Tm-VIG and Tm-IIG, isolated and purified from the venom of Taiwan habu, are shown to have strong anti-clotting and hypotensive effects on experimental animals without being associated with any activity that promote formation of fibrin clots. Tm-VIG is so named because its first three N-terminal residues are Val-Ile-Gly while Tm-IIG has Ile-Ile-Gly at the N-terminalus. The isolation and purification are conducted according to applicant""s previous disclosure in Biochem. Mol. Biol. Int. 31, 1041-1050 (1993). The content of that publication is incorporated herein by reference in its entirety.
Purified Tm-VIG and Tm-IIG show strong beta-fibrinogenolytic activity, cleaving beta-chain of fibrinogen molecules specifically. They also show strong kallikrein-like activity in vitro, releasing bradykinin from kininogen, but do not coagulate human plasma. Thus, they can decrease fibrinogen levels in plasma and prolong bleeding time without causing formation of fibrin clots, indicating that their specificity is different from thrombin and thrombin-like proteases from snake venoms previously known. Furthermore, these two proteases has a high thermal stability than ancrod and thrombin. In rats, intravenous injection of either of the two proteases has the effect of lowering blood pressure. It is noted that these proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the observed in vivo hypotensive effect.
Thus, the present invention discloses a method of using snake venom to treat hyertension in animals and human subject. Although various snake venom components have been isolated and purified, to applicants"" knowledge, no one has taught or suggested the use of a component from snake venom to treat hypertension.
As another object of the present invention, a method of relatively large-scale production of venom proteases useful in hypertension therapy is provided using a protein expression system. Instead of using the conventional enterokinase recognition site, an autocatalytic site is used, based on cleavage specificity of the serine protease, such as TM-VIG and TM-IIG, for post-expression removal of the polyhistidine tag. Genetic engineering is performed so that the autocatalytic site flanks on the 5xe2x80x2-end of the protease gene. Renaturation of the expressed fusion protein showed that the recombinant protease had refolded successfully from the inclusion bodies. Upon autocleavage, the polyhistidine tag with additional amino acid residues appended to the N-terminus of the coding sequence is found to be removed completely. Characterization of the final enzyme produced by the method of the present invention demonstrates that the enzyme is indistinguishable to the one purified from native sources. For example, the recombinant enzyme of the present invention cleaves N-benzoyl-Pro-Phe-Arg-p-nitroanilide, a unique and strict substrate for native Tm proteases reported previously.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawing and descriptive matter in which there are illustrated and described preferred embodiments of the invention.